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Method for detecting avian paramyxoviruses

An avian paramyxovirus and detection kit technology, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. , high sensitivity and high specificity

Inactive Publication Date: 2010-11-24
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the traditional pathogen isolation method is effective and plays an important role in clinical diagnosis, it also has the obvious limitation that it takes a long time to confirm the diagnosis (usually more than a week)

Method used

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  • Method for detecting avian paramyxoviruses
  • Method for detecting avian paramyxoviruses
  • Method for detecting avian paramyxoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The pretreatment of embodiment 1 sample

[0019] 1. Experimental reagents and main instruments

[0020] Main reagents: sterile saline

[0021] Main instruments: Centrifuge 5810R low-temperature desktop centrifuge, a product of Eppendorf, Germany; vortex oscillator; grinder

[0022] 2. Experimental steps

[0023] (1) Tissue sample processing: take 1 mg of organ tissue sample, add 1 ml of sterilized saline, grind and suspend with a grinder, centrifuge the tissue suspension at 3000 rpm for 30 min, and take the supernatant for detection.

[0024] (2) Cloacal or oropharyngeal swab sample processing: add 0.5ml sterilized physiological saline to the swab sample and shake and suspend it with a vortex shaker. The sample suspension is centrifuged at 3000rpm for 30min and the supernatant is taken for detection.

Embodiment 2

[0025] Example 2 Extraction of sample total RNA

[0026] 1. Experimental reagents and main instruments

[0027] Main reagents: Trizol RNA extraction reagent, Invitrogen product, purchased from Beijing Tianyouda Company; DEPC treated water, purchased from Beijing Santai Xinglong Technology Co., Ltd.; new chloroform, isopropanol and absolute ethanol; 75% ethanol; Rnase- free centrifuge tubes and tips

[0028] Main instruments: Centrifuge 5810R low-temperature desktop centrifuge, product of Eppendorf, Germany; biological safety cabinet, product of Forma Scientific

[0029] 2. Experimental steps

[0030] (1) Take 250 μl of the suspension of the sample to be tested, add 750 μl Trizol, invert and mix for 15 seconds until the liquid becomes viscous, and ice-bath for 15 minutes;

[0031] (2) Add 200 μl chloroform, mix by inversion for 15 seconds, and ice bath for 15 minutes;

[0032] (3) The mixture was separated into two phases by centrifugation at 12000 rpm and 4°C for 15 minutes;...

Embodiment 3

[0039] Example 3 Reverse transcription to generate cDNA

[0040] 1. Experimental reagents and main instruments

[0041] Main reagents: reverse transcriptase (Reverse Transcriptase) 200U / μl (Promega); nuclease inhibitor (Rnase Inhibitor) 50U / μl (Takara); 5 times the volume of reaction buffer (5×Reaction Buffer); dNTP mixture 2.5 mM, purchased from Beijing Qiangxin Borui Biotechnology Co., Ltd.; Random Primer (Random Primer) 500 μg / ml (Promega), which is a random hexamer; DEPC-treated water, purchased from Beijing Santai Xinglong Technology Co., Ltd.

[0042] Main instruments: PCR amplification instrument (T-Gradient Thermoblock), purchased from Beijing North Huaao Trading Co., Ltd.; small desktop centrifuge (Eppendorf, Germany); DK-8D electric heating constant temperature water tank (Shanghai Senxin Experimental Instrument Co., Ltd. ).

[0043] 2. Experimental steps

[0044] (1) Add the following ingredients into a 0.2ml centrifuge tube:

[0045] RNA solution 6μl

[0046] ...

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Abstract

The invention provides a method for detecting avian paramyxoviruses, which detects RT-PCR of avian paramyxoviruses (APMV) by using specific primers. The method comprises the following steps of: performing extraction and reverse transcription of total sample RNA to obtain sample cDNA; amplifying target fragments by using the specific primers; performing gel electrophoretic analysis; and judging results. A pair of specific detection primers is designed according to conservative region sequence of NP genes of different serotype avian paramyxoviruses, the sequence is as shown in Seq ID NO:1 and Seq ID No:2, and target gene fragments of 387bp are amplified. The method has the characteristics of high specificity, high flexibility, high efficiency, low cost, suitability for quickly detecting the different serotype avian paramyxoviruses as well as analyzing and detecting a large number of samples and extremely important significance for quickly diagnosing the avian paramyxoviruses at early stage as well as analyzing molecular epidemiology, and simultaneously provides technical means for research on the molecular variation mechanism of the avian paramyxoviruses.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly detecting different serotypes of avian paramyxoviruses by using reverse transcription polymerase chain reaction (RT-PCR) technology. Background technique [0002] Avian paramyxovirus (Avian paramyxovirus, APMV) belongs to the order of monomolecular negative-strand RNA virus (Mononegavirales), paramyxoviridae (Paramyxoviridae), paramyxovirinae (Paramyxovirinae), avian mumps virus (Avulavirus), it includes Nine serotypes, namely APMV-1 (Avian paramyxovirus type-1) to APMV-9 (Avian paramyxovirus type-9). Among them, APMV-1 includes all Newcastle disease viruses, is one of the most important pathogens of poultry, and is also the most comprehensive and in-depth study of avian paramyxoviruses so far. The genome of avian paramyxovirus is a single-stranded negative-strand RNA with a size of about 15kb or 19kb, encoding six main structural proteins, including...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张国中张蕊张守平王晓婷赵继勋
Owner CHINA AGRI UNIV
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